In vitro growth of tissues suitable to the formation of bone and bone forming tissue formed thereby

ABSTRACT

The present invention is directed to a method of growing new bone, bone-like tissue or extracellular matrix under in vitro cell culture conditions. The method utilizes a bioreactor allowing for the flow of nutrient solutions into, through, and out of the bioreactor, wherein ground demineralized bone and bone-forming cells are present in the bioreactor. The resulting bone, bone-like tissue or extracellular matrix produced by the invention are within the scope of the present invention. In addition, the present invention is directed to the bioreactor device used to grow the new bone, bone-like tissue, or extracellular matrix.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/466,772 filed May 1, 2003, expressly incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to the formation of a tissue-engineered materialusing in vitro cell culture, in a bioreactor system(s), in the presenceof biomaterials suitable for the induction of new bone formation. Thisinvention further relates to the use of specific forms of reactors tocause the formation of a shaped material suitable to specific clinicalapplications. For example, the formation of a mandible-shaped reactorfor in vitro growth of a shaped bone graft substitute for the use inrepair of fractured jaws is within the scope of the present invention.This invention further relates to a bone forming tissue that willremodel into load-bearing bone when implanted in the surgical repair ofbone defects.

BACKGROUND OF THE INVENTION

Demineralized bone matrix (DBM) is widely used in the repair ofpathologies associated with skeletal defects and periodontal diseases.This material is typically produced from cortical bone of long-bones(chiefly those bones found in the legs and arms of human cadavericdonors) by cutting the shafts of these long-bones into small chunks (1-4mm) using methods well-known in the field. The resulting pieces andchunks of bone are subsequently cleaned and grinded into a finer bonepowder. The resulting bone powder is typically in the about 125 to 1000micron particle size ranges. The bone powder may be demineralized byexposure to dilute (normally 0.4 to 0.6 N) hydrochloric acid, organicacids, calcium chelating agents, etc. as is known in the art. Forexample, U.S. Pat. Nos. 5,275,954; 5,531,791; 5,556,379; 5,797,871;5,820,581; 6,189,537; and 6,305,379 describe methods of demineralizingbone material and are hereby incorporated by reference in theirentirety. This ground demineralized bone matrix material has been calleddemineralized freeze-dried bone allograft (DFDBA), demineralized boneallograft (DBA), demineralized bone matrix (DBM), and demineralized bone(DMB) and is currently produced by a number of for profit andnot-for-profit companies for use in orthopaedic, spinal fusion, andperiodontal applications.

The use of DBM in the formation of new bone has been assessed using invivo (usually a mouse or rat implant system), in vitro (cell culture orextraction and quantitation of bone forming molecules reportedly presentin bone), and in situ (where the formation of new bone in patients hasbeen assessed during clinical applications) applications. Methods ofassessing this new bone formation and the effects of thedemineralization process on new bone formation by DBM are described inZhang et al., “A quantitative assessment of osteoinductivity of humandemineralized bone matrix.,” J. Periodontol. 68:1076-1084 (1997) andZhang et al., “Effects of the demineralization process on theosteoinductivity of demineralized bone matrix,” J. Periodontol.68:1085-1092 (1997). An in vitro assessment of the ability of DBM toinduce cells towards an osteoblastic phenotype has also been described(Wolfinbarger and Zheng, “An in vitro bioassay to assess biologicalactivity in demineralized bone,” In Vitro Cell Bio. Anim.29A:914-916(1993)).

DBM is assumed to form new bone when implanted in animal models via anendochondral pathway. The implanted DBM is presumed to cause mesenchymalstem cells (typically undifferentiated fibroblasts) to migrate towardsthe implanted biomaterial(s). This induced chemotaxis results in cellsinfiltrating the implanted DBM biomaterial(s) where they are induced toundergo phenotypic changes from a fibroblastic cell phenotype to achondrocyte phenotype and eventually to an osteoblast cell phenotype.These induced phenotypic changes have been reported to be due to theaction(s) of one or more small molecular weight proteins falling in theTGF-β family commonly referred to as bone morphogenetic proteins (BMPs).As the change in cell phenotypes occurs, the proliferative potential ofthe cells declines. For example, the population doubling times increasesfrom approximately 12 hours to approximately 40 hours. As a result, thecells synthesize and secrete collagens and other matrix-formingproteins/glycoproteins laying down a cartilagenous matrix and finally anosteoid-like matrix, which if left implanted in the animal long enough,can be shown to mineralize. This process is analogous to the formationof new bone. If the implanted materials lack the cell-inducing proteinfactors, only providing an environment suitable for cellularinfiltration and cellular proliferation and differentiation, theimplanted materials are deemed to be osteoconductive. If the implantedmaterials possess the cell inducing protein factors and provide anenvironment suitable for cellular infiltration and cellularproliferation and differentiation, the implanted materials are deemed tobe osteoinductive. If the implanted materials already contain cellssuitable for new bone formation, such as autogenously transplanted bone,the materials are deemed to be osteogenic.

SUMMARY OF THE INVENTION

The present invention is directed to a method of growing new bone orbone-like tissue under in vitro cell culture conditions comprisingproviding ground demineralized bone and bone-forming cells in abioreactor under conditions sufficient to form bone or bone-like tissuesuitable for transplantation by causing a flow of nutrient solutionsinto, through, and out of the bioreactor. The bone or bone-like tissueis formed by proliferation and/or differentiation of the bone-formingcells in the presence of the ground demineralized bone and undersuitable bioreactor conditions.

The bone-forming cells are preferably selected from the group consistingof stem cells, fibroblast cells, periosteal cells, chondrocytes,osteocytes, pre-osteoblasts, and osteoblasts. The most preferredbone-forming cells are fibroblast cells and pre-osteoblasts. Thebone-forming cells can be autogenic, allogenic or xenogenic with respectto the intended recipient.

In accordance with the invention, the ground demineralized bone may bein the form of particles or fibers. The particles are about 50 micronsto about 4 mm, preferably about 250 microns to about 710 microns. Thefibers have a width of about 0.1 mm to about 0.5 mm, a thickness ofabout 0.05 mm to about 0.5 mm, and a length of about 1 mm to about 500mm. If the ground demineralized bone is freeze-dried, it should berehydrated. The invention provides that rehydration may occur eitherprior to or after being added in the bioreactor.

The invention further provides that additional components may be addedto the bioreactor, such as collagen or hyaluronin, which may create aviscous bone-like matrix. Additionally, growth factors, such as vascularendothelial growth factor or differentiation factors such as bonemorphogenetic proteins may be added.

The nutrient solution may comprise at least one of Dulbecco's modifiedEagle's medium, fetal bovine serum, L-ascorbic acid-2-phosphate,antibiotics, dexamethasone, beta-glycerolphosphate, glucose, glutamine,amino acid supplements, glutathione-ethyl ester, antioxidants, caspaseinhibitors, and inorganic ions suitable for mineralization-relatedmetabolic events.

The nutrients solution may be delivered to the ground demineralized boneand bone-forming cells by resorbable hollow fibers. The hollow fibersare also sufficient to remove metabolic waste products from thebioreactor.

In another aspect of the invention, nondemineralized bone may be addedalong with the demineralized ground bone. The ratio of demineralizedground bone to nondemineralized bone may be about 1:1 to about 20:1 oras necessary to control availability of biologically active agents andavailable volume for cell growth.

The present invention is further directed to the bone or bone-liketissue formed according to the process disclosed herein. Moreover,implants comprising the bone or bone-like tissue is within the scope ofthe invention.

Furthermore, a method for growing an extracellular matrix capable offorming bone when transplanted into a patient is described. The methodcomprises providing bone-forming cells in a bioreactor under conditionssufficient to promote the growth and differentiation of cells resultingin the formation of an extracellular matrix, wherein said conditionsinclude the flow of nutrient solutions through the bioreactor.Preferably, ground demineralized bone is added to the bioreactor. Thepresent invention further encompasses the extracellular matrix made bythis process and a method of implanting bone into a patient in needthereof comprising transplanting the formed extracellular matrix intothe patient under conditions sufficient to form bone.

In yet another aspect of the invention, a device for the growth of newbone or bone-like tissue under in vitro cell culture conditions isprovided. The device comprises a bioreactor, wherein the bioreactorcomprises inlet and outlet ports for the flow of nutrient solutions,sample injection ports, and an inlet port and outlet port for thebioreactor to cyclically receive negative pressure and positivepressure. The bioreactor may optionally include hollow fibers for thedelivery of nutrients and removal of wastes. The bioreactor is capableof applying mechanical/electrical stimuli to the formed or forming bone.

The bioreactor may further comprise an outer nondeformable chamber andinner deformable chamber. Either of these chambers may receive or removethe nutrient solutions via the inlet and outlet ports. In addition, thesample injection port may contact either chamber in which the bioreactorwill receive biomaterials. Additional ports may be available to allowthe bioreactor to receive cyclical negative and positive pressure in thevolume between the outer nondeformable chamber and the inner deformablechamber through the inlet and outlet ports. Endplates may be used tosecure the bioreactor and provide apertures to receive the ports.

Preferably, the device comprises hollow fibers, which can be in anyshape. The hollow fibers can be round and tubular, or in the form ofconcentric rings. The hollow fibers may be made of a resorbable ornon-resorbable membrane comprising polydioxanone, polylactide,polyglactin, polyglycolic acid, polylactic acid, polyglycolicacid/trimethylene carbonate, cellulose, methylcellulose, cellulosicpolymers, cellulose ester, regenerated cellulose, pluronic, collagen,elastin, or combinations thereof. The pores of hollow fibers are of aspecified diameter that extend from the inside to the outside of thewall of the hollow fiber. For example, the pores may have a diameter ofabout 2 kiloDaltons to about 50 kiloDaltons, preferably about 5kiloDaltons to about 25 kiloDaltons, or alternatively, about 2kiloDaltons to about 15 kiloDaltons.

In accordance with the present invention, the device may include aninner deformable chamber comprising a deformable wall. The deformablecomprising a flexible permeable barrier. The flexible permeable barriermay comprise a resorbable or non-resorbable membrane made up ofpolydioxanone, polylactide, polyglactin, polyglycolic acid, polylacticacid, polyglycolic acid/trimethylene carbonate, cellulose,methylcellulose, cellulosic polymers, cellulose ester, regeneratedcellulose, pluronic, collagen, elastin, or a combination thereof. Inaddition, the inner deformable chamber may further comprises a finemesh. Preferably, the fine mesh comprises sterilizable materials and ismade up of stainless steel, titanium, plastic polymer, nylon polymer,braided collagen, silk polymer, or a combination thereof. The fine meshmay have any suitable pore size range such as, for example, betweenabout 0.1 to about 10 mm, about 1 mm and about 5 mm. The fine mesh maybe on the inner surface of the flexible permeable barrier, outer surfaceor both.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a broad overview of a suitable hollow fiberbioreactor system to be used in the in vitro growth of tissue suitableto the formation of bone and bone forming tissue formed thereby.

FIG. 2 illustrates the nutrient delivery and waste removal via hollowfibers of a suitable bioreactor within the scope of the presentinvention.

FIG. 3 illustrates “bone plug” formation in a bioreactor filled with DBMand cells.

FIG. 4 depicts a cross section of a hollow fiber bioreactor within thescope of the present invention, which assists in the calculation of thenumber of hollow fibers for one bioreactor.

FIG. 5 depicts another suitable hollow fiber bioreactor within the scopeof the present having an inner deformable chamber.

FIG. 6 depicts femoral head formation in a hollow fiber bioreactor madeaccording to the method of the present invention.

FIGS. 7A and 7B depict representative “bone plugs” generated in thehollow fiber bioreactor of the present invention. The dashed lines areintended for illustration purposes only. The histological analysis ofthese representative “bone plugs” was further depicted in FIGS. 11 to14.

FIGS. 8A-D illustrate representative “bone plugs” generated in thebioreactor that are subsequently freeze-dried. The shapes of the “boneplugs” reflect the shape of the deformable inner vessel of thebioreactor.

FIG. 9 illustrates the time course of the osteocalcin levels (ng/tube)for different cell seeding densities (0.5, 1.0, 2.0, and 5.0 millionfibroblast cells per 100 mg of DBM) over an incubation period of 7weeks.

FIG. 10 illustrates the osteocalcin levels (ng/ng DNA) for various cellseeding densities (0.5, 1.0, 2.0, and 5.0 million fibroblast cells per100 mg of DBM) on the 2^(nd), 3^(rd), 4^(th), 5^(th), and 6^(th) week ofincubation.

FIGS. 11A, B, and C illustrate the histological analysis of a “boneplug” generated in a bioreactor according to the method of the presentinvention at 200× magnification. The “bone plug” generated in bioreactorwas embedded and sectioned. The sections were stained with the AlizarinRed (FIG. 11A), H&E (FIG. 11B), and Masson's Trichrome (FIG. 11C)methods. The Alizarin Red staining revealed the calcium deposition innewly formed extracellular matrix. H&E staining revealed the changes infibroblast morphology and new extra-cellular matrix (ECM) productionthat appeared to be “osteoid” formation. Masson's Trichrome stainingsuggested that the newly formed extracellular matrix containedsignificant quantities of collagen.

FIG. 12 illustrates the histological analysis of a “bone plug” generatedin a bioreactor according to the method of the present invention at 400×magnification. The sections were stained with the Alizarin Red (FIG.12A), H&E (FIG. 12B), and Masson's Trichrome (FIG. 12C) methods. TheAlizarin Red staining revealed the calcium deposition in newly formedextracellular matrix. H&E staining revealed the changes in fibroblastmorphology and new extra-cellular matrix (ECM) production that appearedto be “osteoid” formation. Masson's Trichrome staining suggested thatthe newly formed extracellular matrix contained significant quantitiesof collagen.

FIGS. 13A-13B illustrate the H&E staining of a “bone plug” generated ina bioreactor according to the method of the present invention and FIGS.13C-13D illustrate the H&E staining of an analogous “bone plug”generated from heterotopic implantation of DBM in a nude mouse (400×magnification). The new bone growth in a bioreactor after 4 weeksincubation was compared to the new bone growth in a nude mouse 4 weeksafter DBM implantation. The changes in fibroblast morphology and newextracellular matrix production appeared on samples.

FIG. 14A illustrates the Mason's Trichrome staining of a “bone plug”generated in a bioreactor according to the method of the presentinvention and FIG. 14B illustrates an analogous “bone plug” generatedfrom heterotopic implantation of DBM in a nude mouse (400×magnification). Significant amounts of new extracellular matrix wereproduced around cells and stained as collagen fibril for both “boneplug” generated in a bioreactor and explants from a nude mouse.

FIG. 15 depicts a graph of the alkaline phosphatase (nmol pNP/min/μg)activity for “bone plugs” generated in a hollow fiber bioreactor withvarious cell seeding densities (0.5, 1, 5, and 10 millions humanperiosteal cells per 500 mg of DBM).

FIGS. 16A and 16B illustrate the H&E staining for a “bone plug”generated in a hollow fiber bioreactor (400× magnification) according tothe method of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural references unlessthe context clearly dictates otherwise. Thus, for example, reference to“a nutrient solution” includes a plurality of such solutions andreference to “the vessel” includes reference to one or more vessels andequivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,or constructs similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices, or constructs are now described.

The term “bioreactor” is intended to mean a contained or enclosed systemor vessel for the culture of cells, such as mammalian or vertebratecells, by which sterility or the freedom from microbial contaminationcan be achieved. Nutrient solutions can be aseptically delivered intothe bioreactor and waste solutions can be aseptically removed from thebioreactor.

The term “newly formed bone” is intended to mean a matrix secreted bybone-forming cells. This newly formed bone is best illustrated byhistological evidence of newly formed bone when demineralized bone isimplanted intermuscularly in a nude mouse (or rat) bioassay system. Forexample, FIG. 12 depicts new bone growth in a bioreactor within thescope of the present invention.

The term “bone tissue” is intended to include the organic phase ororganic and inorganic phases of that tissue comprising a bone. Withinthe context of this invention, bone tissue can include newly formedbone, implant bone, and associated cells, bone marrow, bone marrow-liketissue, and cartilage (and cartilage-like tissues).

The term “bone-like tissue” is intended to include a matrix similar tocartilage and/or osteoid similar to that tissue found in articularcartilage, mineralized adult bone, nonmineralized fetal bone, or tissuesconsisting primarily of type 1, type 2 collagens, hyaluronic acid(hyluronans), proteoglycans, and non-collagenous proteins similar tothose proteins found in bone and/or cartilagenous tissues. This matrixwill be suitable for the growth and differentiation of chondrocytes,chondrocyte-like cells, osteocytes, osteoblasts, and/or osteoblast-likecells.

The term “transplantable bone” is intended to include a nonmineralized,partially mineralized, or fully mineralized viable construct produced,using a bioreactor, that is nonload-bearing, partially load-bearing, orfully load-bearing at the time of transplantation.

The term “implantable bone” is intended to include a nonmineralized,partially mineralized, or fully mineralized nonviable acellularizedconstruct produced, using a bioreactor, that is nonload-bearing,partially load-bearing, or fully load-bearing at the time ofimplantation.

The term “strain” is intended to include forces applied to the cells andmatrix contained in a bioreactor that contribute to manipulation ofphenotype of the cells contained therein. As used in the presentinvention, strain is expected to be applied to the cells and matrix inthe bioreactor through forces applied to and within the bioreactor.

The term “stress” is intended to include forces applied to the cells andmatrix contained in a bioreactor that contribute to manipulation ofphenotype of the cells contained therein. As used in the presentinvention, stress is expected to be applied to the cells and matrix inthe bioreactor through forces applied to and within the bioreactor.

The term “hollow fiber” is intended to include tubular structurescontaining pores of defined size, shape and density for use indelivering nutrients (in solution) to cells contained within abioreactor and for removal of waste materials (in solution) from cellscontained within a bioreactor. For purposes of the present invention,hollow fibers may be constructed of a resorbable or nonresorbablematerial.

The term “nutrient solution” is intended to include solutions entering abioreactor and containing those nutrient materials essential to theculture of mammalian or vertebrate cells. Nutrient solutions may alsocontain additives that affect specific changes in phenotype of cellsunder culture or to contribute to changes in the matrix structure of theforming newly formed bone, such as, mineralization.

The term “waste solution” is intended to include solutions exiting abioreactor and containing waste byproducts of cellular metabolism. Theconcentrations of waste byproducts, for example ammonia, lactic acid,etc. and residual levels of nutrients such as glucose, in the wastesolution can be used to assess the levels of metabolic activity of cellsbeing cultured in a bioreactor.

The present invention provides a method of growing bone in vitroinvolving providing a biomaterial, such as ground demineralized bone,suitable for inducing cells to form an extracellular matrix and cellscapable of forming bone or bone-like biomaterials, and placing thebiomaterial and bone-forming cells in close association under conditionssuitable for forming bone or bone-like biomaterial. In particular, theground demineralized bone and bone-forming cells are preferably placedin a bioreactor capable of simulating the nutrient flow and wasteremoval present within an implant site. The flow of nutrient solutionsinto, through, and out of the bioreactor permit the associated grounddemineralized bone and bone-forming cells to form into bone or bone-likebiomaterial suitable for transplantation.

The biomaterial, ground demineralized bone, is capable of inducingselected cell types to form an extracellular matrix consistent with theosteoid materials comprising the organic phase of bone tissue whenimplanted in heterotopic or orthotopic sites in a living organism.Ground demineralized bone is obtained in manners known in the art andmay be available in any form, including as particles or fibers. Grounddemineralized freeze-dried bone particles may be used in any particlesize suitable for inducing the growth of bone in a bioreactor, such asfrom about 50 microns to 4 mm, preferably, about 125 microns to 850microns, and most preferably, about 250 microns to 710 microns. Grounddemineralized bone fibers may be produced in known manners, such as byskiving or shaving the surface of the cortical bone to produce shortfibers that easily entangle. The fibers are suitable for growing bone ina bioreactor and preferably have physical dimensions of about 0.1 mm to0.5 mm in width, 0.05 mm to 0.5 mm in thickness, and 1 mm to 500 mm inlength. The bone used to make the ground demineralized bone may beprocessed in known manners prior to forming the ground demineralizedbone used in connection with the present invention. For example, thebone may be treated with enzymes to partially digest the organiccomponents of the bone, such as collagenase, papain, protease,hyaluronidase, endonuclease, lipase, and/or phosphatase, or organicacids, such as acetic or citric acid. Alternatively, the bone may bepartially digested by fragmenting the covalent bonds in the individualcollagen molecules contained in the demineralized bone. The covalentbond breakage of the formed fragments of a collagen molecule may be inthe range of about 2 to about 50, and should be sufficient to modify theresorption rate of the demineralized bone. Subsequent to forming thefibers or particles, the fibers and particles are demineralized byexposure to dilute (about 0.4 to 0.6 N) hydrochloric acid or organicacids, calcium chelating agents, etc., as one skilled in the art wouldappreciate. Alternatively, non-acid chelators of calcium, such asethylene diamine tetraacetic acid (EDTA), may be used to demineralizethe bone.

In addition, the weight percent residual calcium in ground demineralizedbone is a factor in defining the bioavailability of bioactive molecules,such as, for example, bone morphogenetic proteins (BMPs), to thecellular population contained within the bioreactor. In fact, it hasbeen found that the ability to extract BMPs from ground bone particleshas been shown to be approximately a linear function of the extent ofdemineralization of the ground bone. Thus, a suitable amount of residualcalcium is that amount sufficient to optimize the bioavailability ofbioactive molecules, such as BMPs, to the bone-forming cells in thebioreactor. Preferably, the residual calcium is present in the range ofabout 0-8 weight percent, more preferably about 1-4 weight percent, andmost preferably about 2 weight percent.

In accordance with the present invention, the ground demineralizedfreeze-dried bone particles are added aseptically to the bioreactor.They may be directly added to the bioreactor in a freeze-dried state andrehydrated in the bioreactor or rehydrated in culture medium prior toaddition to the culture chamber of the bioreactor. The grounddemineralized bone may be added alone or in combination with othercomponents. Preferably, the other components do not inhibit the effectof the ground demineralized bone to induce bone formation. For example,ground nondemineralized bone may be added with ground demineralizedbone. In such cases, the ground demineralized bone to nondemineralizedbone may be added in any ratio, but preferably is added in a ratio ofabout 1:1 to about 20:1, more preferably about 8:1, and most preferablyabout 3:1. The ground nondemineralized bone may take any form, e.g.,particles or fibers, and typically will have similar physical dimensionsas the ground demineralized bone.

Particle size ranges of the ground demineralized bone particles in thebioreactor determine the “void volume” or available volume outside ofthe ground demineralized bone particles in which the bone-forming cellsand other components may be added. It has been found that the boneparticle spacing or availability of space around the grounddemineralized bone particles within the bioreactor relates to the voidvolume and has an impact on the ability of bone-forming cells in thebioreactor to differentiate and/or proliferate. It is desired thatbone-forming cells have sufficient contact to allow those cells toinfiltrate the voids or space between the ground demineralized boneparticles, which permits the in vitro growth of bone or bone-liketissue. Therefore, the void volume or spacing around the grounddemineralized bone particles should be that which is effective inallowing for the optimal contacting and infiltration of voids bybone-forming cells between the ground demineralized bone particles.

In accordance with the present invention, the ground demineralized boneparticles may be rehydrated in the bioreactor or prior to being added tothe bioreactor. Preferably, the particles are rehydrated and mixed withbone-forming cells prior to addition to the bioreactor. The grounddemineralized bone particle spacing will differ depending on whether ornot the bone particles are rehydrated prior to addition to thebioreactor growth chamber. First, the ground demineralized boneparticles may be added to the bioreactor growth chamber and subsequentlyrehydrated prior to adding bone-forming cells. In this approach, theground demineralized bone particles may be added to the bioreactorgrowth chamber in a freeze-dried state, which provides a relativelysimple step and allows the particles to pack tightly filling theavailable space. Subsequent rehydration of these freeze-dried grounddemineralized bone particles in the bioreactor will cause the boneparticles to swell to a tighter state of packing due to rehydration. Thebone-forming cells may then be added to the rehydrated bone matrix voidvolume (that volume outside of the bone particles) in the bioreactor. Ithas been found this tighter state of packing ground demineralized boneparticles in the bioreactor is effective in more tightly packing theadded bone-forming cells. While the tight packing may hinder someinfiltration of the void volume present throughout the bioreactor, ithas been found that the more tightly packed added cells promotes betterretention of synthesized matrix molecules during the differentiationprocess and may be best utilized when seeding more differentiated cellsinto the bioreactor system.

Alternatively, the ground demineralized bone particles may be rehydratedprior to the addition to the growth chamber of the bioreactor. Thebone-forming cells may then be added to the packed ground demineralizedbone particles in the bioreactor or directly to the rehydrated boneparticle suspension prior to its addition to the bioreactor. Whilerehydrating freeze-dried ground demineralized bone particles prior toaddition to the growth chamber of the bioreactor has been found toincrease the difficulty in adding the bone particles to the bioreactor,it has been found that directly adding the bone-forming cells to therehydrated ground demineralized bone particle suspension results infully dispersed bone-forming cells and ground demineralized boneparticles. More uniform distribution within the growth chamber isthereby achieved and is less likely to contribute to damage to thehollow fibers present within the growth chamber.

In either case, centrifugal forces can be used to cause the rehydratedbone particles and cells to pack throughout the growth chamber withexcess fluids removed from the packing port.

The “bone-forming cells” of the present invention are those cellssuitable for the induction of new bone formation when infiltrated withground demineralized bone in a bioreactor and include those cell typessuitable for differentiating into bone cells or suitable for forming amatrix similar to osteoid of forming new bone. Suitable cell typesinclude, but are not limited to stem cells, fibroblast cells, periostealcells, chondrocytes, osteocytes, pre-osteoblasts, and osteoblasts.Preferably, the stem cells are multipotent, the fibroblast cells areundifferentiated, the periosteal cells are partially differentiated, andthe chondrocytes or osteocytes are differentiated. In the case ofdifferentiating cell types, such as fibroblasts or stem cells, thesecell types may be placed in close proximity to the ground demineralizedbone, which, in the bioreactor and under appropriate conditions, willcause the cells to differentiate into bone cells. In the case of celltypes suitable for forming an osteoid-like matrix, such as osteoblastsor chondroblasts, such cell types may be placed in close proximity tothe ground demineralized bone in the bioreactor and under appropriateconditions, will cause the cells to synthesize matrix similar to osteoidof forming new bone. The type of cells selected for in vitro bone growthis dependent upon the desired time frame for new bone formation, seedingcell densities, and nutrient medium provided.

The source of the bone-forming cells may be autogenic, allogenic, orxenogenic. The use of a potential recipient's own cells in the formationof the bone or bone-like biomaterial will result in a tissue unlikely tobe rejected for some immunological reason, rendering the transplantablenewly formed bone autogenous in nature. The use of allogenic cells inthe formation of new bone with subsequent implantation can be achievedby decellularizing any newly formed bone or bone-like structure prior toimplantation using any decellularizing technology known in the artdepending on the desired characteristics of the acellular bone orbone-like structure desired for a given clinical application.

The bone-forming cells are added either to the void volume space of thepacked ground demineralized bone particles or directly to the rehydratedground demineralized bone particles prior to addition to the growthchamber of the bioreactor. The cell density of the bone-forming cellsmay be in the range of from about 10²-10⁸ cells per ml, preferably10³-10⁶ cells per ml, and more preferably about 10⁴-10⁵ cells per ml.The density of bone-forming cells added depends on several factors. Forexample, previous cell culture work in development and validation of invitro bioassays for assessing the osteoinductive potential ofdemineralized bone demonstrated the importance of cell densitydifference depending on the phenotypic status of the cells.(Wolfinbarger, L and Y. Zheng. 1993. An in vitro bioassay to assessbiological activity of demineralized bone. In Vitro Cell Dev. Biol.Anim. 29:914.) Less differentiated cells (e.g., dermal fibroblasts),where proliferation constituted a component of the differentiationprocess, involved a lower seeding density in in vitro bioassays thanmore differentiated (periosteum derived cells, for example) cells.Presumably, cells more differentiated along the pathway leading from a“stem-like” cell to a differentiated cell phenotype proliferated lesswell (longer population doubling times of approximately 40 hours) thanless differentiated cells (shorter population doubling times ofapproximately 12 hours) and could be seeded at higher cell densitieswhen used in an in vitro bioassay. Consequently, seeding densities ofcells in the bioreactor depends in part on the phenotype of the cellsadded to the bioreactor, the availability of biologically activematerials, and the culture medium used. In addition, seeding celldensity in the bioreactor depends on the ability to deliver nutrients tothe cells and remove waste byproducts from the bioreactor culturechamber. For example, greater cell densities in the bioreactor requiremore nutrient delivery and greater waste product removal than lower celldensities.

The bioreactor can be in virtually any shape based on the shape of thebioimplant desired as a newly formed bone or structure that will formload-bearing bone when implanted clinically. The wall of the bioreactorcan be deformable and contained within a nondeformable chamber such thatpositive and negative pressure environments can be applied between theinner wall of the nondeformable chamber and the outer wall of thedeformable chamber such that the volume of the bioreactor containing thedemineralized bone, cells, and matrix can be decreased or increased overtime to simulate stress and strain application to the bone matrix beingformed.

The demineralized bone and bone-forming cells can be preloaded into thebioreactor in the presence, or lack thereof, of a viscous matrixdesigned to provide attachment sites for the cells and/or to restrictdiffusion of synthesized osteoid forming molecules. The viscous natureof the matrix may be obtained by the incorporation of polymers, forexample, collagenous, hyaluronin, or similar resorbable or nonresorbablepolymers.

Nutrients are delivered to the ground demineralized bone andbone-forming cells in the bioreactor and may impact the growth anddifferentiation of cells contained in the bioreactor. The nutrientsolutions are selected to provide sufficient nutrition to thebone-forming cells to maintain viability, growth, and/or differentiationin the bioreactor. Those skilled in the art are capable of selecting anappropriate nutrient solution for the present invention. For example,media such as Dulbecco's modified Eagle's medium may be used and may befurther supplemented with other suitable nutrients. Other suitablenutrients include fetal bovine serum, L-ascorbic acid,-2-phosphate,antibiotics, cell modulators such as dexamethasone,beta-glycerolphosphate, glucose, glutamine, amino acid supplements,inhibitors (or activators) of apoptosis such as glutathione-ethyl ester,antioxidants, caspase inhibitors, and cations and anions, e.g.,magnesium, manganese, calcium, phosphate, chloride, sodium, potassium,zinc, and sulfate ions, and nitrates and nitrites. The concentration offetal calf serum must not inhibit induced cell differentiations due todiffusible agents from the demineralized bone. The remainingconcentration of components in the nutrient solution should besufficient to promote growth and/or differentiation in the bioreactorand maintain viability of the bone-forming cells and the resulting boneor bone tissue.

In accordance with the present invention, the nutrient solutions may bemodified during different phases of the process. For example, duringinitial culture, seeded cell densities may be minimal, especially forfibroblast cell seeding cultures, and thus nutrient solutions maycontain low concentrations of fetal calf serum (such as ≦2% vol:vol) tofacilitate the role of growth and differentiation factors diffusing fromthe ground demineralized bone particles in modulating phenotypic changesin the added cells. Monitoring the concentration of the nutrients, suchas glucose, glutamine, and amino acid supplements, via the eluent flowof medium allows for the determination of nutrient consumptionpermitting control of flow (delivery) of nutrients into the cellpopulation. Moreover, waste products of metabolism, for example, ammoniaand lactic acid, can be monitored via the eluent flow of medium from thebioreactor to determine the metabolic state/function of the residentcell population. Changes in cell phenotype during the culture phase canbe monitored by sampling the eluent flow of medium from the bioreactorfor proteins associated with specific cell phenotypes, for example,osteopontin and osteocalcin. Should it be desired, for example, othercomponents may be added to the medium during culture to promote adesired function. For example, to induce mineralization during aspecific phase of the culture period, chemical components such asβ-glycerolphosphate may be added to the medium as a substrate foralkaline phosphatase and to serve as a source of phosphate to becomplexed with calcium in the formation of crystalizable calcium saltssuch as hydroxyapatite. Alternatively, hormonal stimulation of cells canbe accomplished via the addition of certain compounds such as, forexample, vitamin D. The levels of oxygen tension can be controlled byoxygenation of the nutrient medium being added to the cells beingcultured in the bioreactor to manipulate the metabolic state of thecells during the culture phase such that mildly hypoxic conditions canbe used to manipulate chondrogenesis and/or osteogenesis. Manipulationof the ionic composition of the medium can be used to control hydrolyticenzyme degradation of demineralized bone matrix, enzyme mediatedcross-linking of the formed extracellular matrix being synthesized bythe resident cell population, and the osmotic balance of the nutrientsolution. Induction and/or inhibition of cellular apoptosis can becontrolled by the addition of inhibitors (or activators) of apoptosissuch as glutathione-ethyl ester, antioxidants, and caspase inhibitors oractivators. For example, use of allogenic cells may require induction ofapoptosis to produce acellular formed bone tissue. In addition, gammairradiation treatment of the bone particles, either before or afterdemineralization, can be used to promote cell-mediated resorption of thedemineralization bone particles facilitating new bone formation withinthe areas where the bone particles are resorbed.

The nutrients may be delivered in any manner suitable for the formationof bone in the bioreactor. For example, resorbable hollow fibers can beused to deliver nutrients and remove metabolic waste products during thecellular proliferations and/or differentiation process. The nutrientsolutions used can be sequentially introduced into the bioreactor growthchamber as needed to induce cellular morphogenesis, growth, secretion ofosteoid biomaterials, and/or to cause mineralization of the formedmatrix as desired depending on the type of implantable bone materialdesired. The resorbable hollow-fibers used to deliver nutrients andremove wastes from the bone forming part of the bioreactor provide anopportunity to leave a series of hollow tube-like openings within theformed bone tissue through which the formed bone tissue can bevascularized. Growth factors such as vascular endothelial growth factor(VEGF) can be final delivered through these hollow fibers once the bonetissue has been formed to promote angiogenesis within the hollowstructures following transplantation.

Delivery of nutrients and removal of waste products depends primarily ontwo factors: numbers of hollow fibers per unit volume of the culturechamber of the bioreactor and flow rates of nutrient solutions throughthe hollow fibers.

The hollow fibers of the present invention are those suitable for thedelivery of nutrients and removal of waste in the bioreactor. The hollowfibers may be any shape, for example, they may be round and tubular orin the form of concentric rings. The hollow fibers may be made up of aresorbable or non-resorbable membrane. For example, suitable componentsof the hollow fibers include polydioxanone, polylactide, polyglactin,polyglycolic acid, polylactic acid, polyglycolic acid/trimethylenecarbonate, cellulose, methylcellulose, cellulosic polymers, celluloseester, regenerated cellulose, pluronic, collagen, elastin, and mixturesthereof. Moreover, the hollow fibers of the present invention includepores to allow the nutrients and waste to pass in and out of it. Thepores of the hollow fibers are a sufficient diameter to allow thediffusion of a molecule from one side of the hollow fiber to the otherside of the hollow fiber. Preferably, the molecules that may passthrough the hollow fiber pores are about 0.002 to about 50 kDa, morepreferably about 5-25 kDa, or most preferably 2-15 kDa.

The number of hollow fibers per unit volume of the culture chamber ofthe bioreactor is determined based on the cross-section of the hollowfibers, the bioreactor per se, and the distance the bone-forming cellscan live from the hollow fibers for nutrient delivery and waste removal.As an example of determining the number of hollow fibers per unitvolume, FIG. 4 illustrates the cross section of a hollow fiberbioreactor. Assume the bioreactor cross section inner diameter (ID) is 2cm (A), one hollow fiber ID is 1 mm (B), and the distance of cells canlive from any conduit for nutrient delivery and waste removal is 20 μm,the ID of the circular area where nutrient deliver and waste remove byone hollow fiber (C) should equal to B+20×2 μm. Thus the number ofhollow fibers needed for bioreactor can be calculated as follows:

1) Bioreactor ID (A)=2 cm

2) Hollow Fiber ID (B)=1 mm

Distance of Cells Can Live From Any Conduit for Nutrients Delivery andWaste Removal approximates 20˜30 μm depending on the diffusion rates ofthe nutrient molecules. According to human physiology, it is rare thatany single functional cell of the body is more than 20-30 μm away from acapillary.

3) Calculation:

Total  Area  of  Cross-section  of  Bioreactor = (A/2)² * π = (2cm/2)² * π = (10  mm)² * π = 100  mm² * π

Total  Area  of  Cross-section  of  One  Hollow  Fiber = (B/2)² * π = (1  mm/2)² * π = 0.25  mm² * π

-   -   Total Area of Nutrients Delivery and Waste Removal of One Hollow        Fiber=(C/2) ²*π=(1 mm/2+0.02 mm)²*π=(0.5 mm+0.02 mm)²*π=0.2704        mm²*π    -   Number of Hollow Fibers for Bioreactor with Cross-Section ID of        2 cm=100 mm²* π/0.2704 mm²*π=369.82≈370    -   Percentage of Total Area Covered by Hollow Fibers=(0.25        mm²*π)*370/100 mm²* π*100=92.6%    -   Percentage of Total Area Covered by Nutrients Delivery and Waste        Removal=(0.2704 mm²*π)*370/100 mm²*π*100=100.48%

Although the flow of nutrient solutions through the hollow fibers willgenerate some minimal turbulent flow of solutions through the bulkvolume of the growth chamber of the bioreactor, the primary mechanismfor nutrient dispersal through the growth chamber and to the cells inculture will be diffusion and/or the alternating positive and negativepressure applications applied to the deformable bioreactor wall used toapply stress/strain to the demineralized bone, cells, and extracellularformed/forming matrix mixture during the culture process. Diffusion ofnutrients from capillary beds in tissue typically limits the provisionof nutrients (for example oxygen, glucose, etc.) to 20-30 μm from anindividual capillary. Thus, if diffusion were the sole determinant ofnutrient delivery and waste removal, it should be expected that cellslocated more than 20-30 μm from a hollow fiber will receive lessnutrients and exist in a greater concentration of waste byproducts thancells close to a hollow fiber. With application of stress/strain to thedemineralized bone, cells, and extracellular formed/forming matrixmixture via alternating applications of positive and negative pressure,it becomes possible to affect greater nutrient solution delivery andwaste removal permitting cultivation of cells at greater distances fromthe hollow fibers than would be allowed by simple diffusion.

Shear stress to cells present in the bioreactor due to flow of nutrientsolution will be minimal. Thus, optional addition of mechanical stressand strain to the forming bone matrix will occur primarily viamanipulation of the inner vessel in the bioreactor used to contain thedemineralized bone, cells, and extracellular formed/forming matrix. Thiscomponent of the bioreactor includes the option of placing an innervessel constructed of a deformable material within an outer vessel towhich cyclic positive and negative pressure can be applied via a port inthe outer vessel wall. It is to be expected that such positive andnegative pressures will be minimal and designed to gently compress andexpand the forming extracellular matrix in order to provide cyclicmechanical stimulation to the cells contained within the inner vessel ofthe bioreactor and to promote nutrient solution flow into, through, andout of the bioreactor containing the cells and matrix mixture.

In addition to the cyclic mechanical stimulation to cells containedwithin the inner vessel of the bioreactor, the inclusion of a series ofmicro-electrodes within the inner wall of the inner vessel in liquidcontact with the forming, or formed, extracellular matrix will allowcyclic, low-level, electrical stimulation of cells and/or the creationof a small electrical gradient from one end to the other end, or side toside, of the bioreactor for use in electrical stimulation of cellularmetabolism during induced new bone formation. This cyclic electricalstimulation can occur concurrent with, or not concurrent with, othermechanical or media changes to the forming, or formed, extracellularmatrix containing the cells being manipulated to form new bone orbone-like tissue(s).

One aspect of the present invention is practiced by sterilizing allaspects of the bioreactor (tubing, fittings, valves, reagent (solution)containers, filters, sampling ports, bioreactor components, etc.).

The bioreactor 100 as shown in FIG. 1 illustrates an example of a hollowfiber bioreactor system of the present invention. The bioreactor 100 asset forth in FIG. 1 is aseptically assembled such that the hollow fibers120 are connected to the inlet end-plate 106 and drawn through thetubular vessel 103 of the bioreactor 100 allowing the tubular vessel 103of the bioreactor to be attached to the inlet end-plate 106 forming awater-tight seal. The non-connected end of the hollow-fibers 120 is thencarefully attached to the outlet end-plate 102 forming a water-tightseal. Once the bioreactor is assembled, the ground demineralized bonecan be rehydrated, if not already done so, and cells added via theinjection ports 104 and 105. The bioreactor 100 is attached to at leastone inlet port 107 and at least one outlet port 101 and the flow ofnutrient solution from the nutrient reservoir 112 through the hollowfibers is initiated. The nutrients are delivered from the nutrientreservoir 112 through a noncytotoxic and nonhemolytic tubing 115connected to the outlet port of nutrient reservoir 113 and the inletport of the bioreactor 107. The flow is initiated and maintained inmanners known in the art, but is preferably conducted centrifugal forcesor a pump 114, such as a peristaltic pump, sufficient to cause the flowof media and waste products through the bioreactor 100. A pump 114 ispreferably used to control the flow rate of the nutrients. Initiation offlow of nutrient solutions is important in that the cells contained inthe bioreactor are labile to nutrient deprivation and thus the timebetween addition of cells to the bioreactor and initiation of nutrientsolution flow should not exceed a time in which the specific cellpopulation in the nutrient solution used to pack them becomes depletedof nutrients or changes pH to an extend that the cells becomemetabolically stressed. Additional reagents may added through a reagentaddition port 109 as described above. Moreover, the waste generated fromthe bioreactor is removed through a tubing 108 connected to the outletport 101 of the bioreactor 100 and the inlet port 110 of the nutrientreservoir 112. The eluent of medium from the bioreactor may be monitoredto assess for proteins associated with bone formation, waste products,and nutritional capacity of the cells and demineralized bone, asdescribed. The medium may also be recycled and recirculated into thenutrient reservoir 112 through a recycling inlet port 110. The nutrientsolution in the nutrient reservoir 112 may be changed through thereagent addition port 109. One skilled in the art would appreciate whenthe nutrient solution should be changed. Preferably, the nutrientsolution is changed at least once a week.

Referring to FIG. 2, the nutrient delivery and waste removal via hollowfibers of a bioreactor 200 of the present invention is depicted. Thebioreactor 200 is aseptically assembled such that the hollow fibers 215are connected to the inlet end-plate 210 and drawn through the tubularinner chamber 208 of the bioreactor allowing the tubular inner chamber206 of the bioreactor 200 to be attached to the inlet end-plate 210forming a water-tight seal. The ground demineralized bone is added intothe inner-most volume of the inner vessel 206 before or followingrehydration until it fills the inner-most volume. If the grounddemineralized bone is rehydrated prior to or concurrent with theaddition to the inner-most volume, it is mixed with the cells to be usedat an appropriate seeding density, i.e. number of cells/unit volume ofextra-particle space. If the ground demineralized bone is not rehydratedprior to addition to the inner-most volume, the bone will need to berehydrated prior to addition of cells once the bioreactor is fullyassembled. The non-connected end of the hollow-fibers 215 is thencarefully attached to the outlet end-plate 205 forming a water-tightseal. This inner chamber 206 is now ready for insertion into the outerchamber 209 component of the bioreactor 200. This is accomplished bysliding the outer most diameter of one of the end-plates 211 through theinternal lumen of the outer chamber 209 until the remaining end-plate203 can form a water-tight seal with the inner diameter of the outerchamber 209. As an alternative method, the assembled inner chamber 206can simply be inserted into the outer chamber 209 by guiding (pressing)the end-plates, 203 and 211, into the guide holes present in the innerfaces of the outer chamber 209. Once the bioreactor is assembled, theground demineralized bone can be rehydrated, if not already done so, andcells 214 may be added via the injection ports, 202 or 204. The flow ofthe nutrients would enter via at least one inlet port 212 and exitthrough at least one outlet port 201.

The deformable wall of the inner chamber of the bioreactor may beconstructed out of a flexible permeable barrier and a fine deformablemesh that can be molded to a specific shape as needed. The flexiblepermeable barrier is mechanically supported by a fine mesh, which ispresent either on the inside or the outside of the flexible permeablebarrier. The flexible permeable barrier is made of any suitableresorbable or non-resorbable membrane, such as those comprisingpolydioxanone, polylactide, polyglactin, polyglycolic acid, polylacticacid, polyglycolic acid/trimethylene carbonate, cellulose,methylcellulose, cellulosic polymers, cellulose ester, regeneratedcellulose, pluronic, collagen, elastin, or mixtures thereof. The finemesh is suitably made up of sterilizable materials, such as stainlesssteel, titanium, plastic polymer, nylon polymer, braided collagen, andsilk polymer, but must be capable of deforming to any desired shape. Thefine mesh may -have any suitable pore size dictated by the desired boneplug properties. For example, suitable pore sizes for the mesh isbetween about 0.1 to 10 mm and, preferably, 1-5 mm. The deformable wallmay be made to be permeable for some metabolites and not others. Forexample, the deformable wall may be made to not be permeable to small orlarge molecular weight metabolites. In particular, a small molecularweight metabolite would fall within the range of 0.001 -25 kDa,preferably 0.1-2.5 kDa. A larger molecular weight metabolite would fallwithin the range of 25-200 kDa, preferably 25-50 kDa. The deformablewall may further be constructed to allow for its use in the bioreactorof the present invention. For example, the tensile properties of thedeformable wall should make it capable of deforming under the cyclicnegative and positive pressure, such as between 10-30mmHg. The mesh usedto construct the deformable wall preferably will conduct an electricalcurrent. The resorbable or non-resorbable hollow fibers can be used todeliver nutrients and remove waste for the inner chamber. The deformableinner chamber can be contained within a nondeformable outer chamber. Thecyclic application of positive and negative pressures to the deformablewall of the inner chamber of the bioreactor to be used in the in vitrogrowth of bone or bone-like tissue serve to transform this bone orbone-like tissue into bone following transplantation into a recipient.

Inlet and outlet ports of the outer chamber can deliver nutrients andremove waste for this deformable chamber (FIGS. 5 and 6). For example,FIG. 5 illustrates a hollow fiber bioreactor with an inner deformablechamber 509, wherein the deformable wall is comprised of a flexiblepermeable barrier 507 and a fine mesh 506. The bioreactor may containone outer chamber 510 and one inner deformable chamber 509. The outerchamber 510 is closed by two end-plates 504 and 511 by means suitablefor closing the chamber, such as an annular groove. The inner deformablechamber 509 is closed by one end-plate 505. The flexible permeablebarrier 507 (non-resorbable membrane or resorbable membrane) and a finemesh 506 are sealed to the plate 505 of the inner deformable chamber509. The inner deformable chamber 509 can be deformed to the desiredshape using a deformable metal mesh 506. At least one inlet 512 and atleast one outlet 502 port is connected to the outer chamber 510 and areused for the nutrient delivery and waste removal in the outer chamber510. Nutrient delivery and waste removal in the inner chamber 509 employthe use hollow fibers 508 connected to the at least one inlet 501 and atleast one outlet 503 port on the outer chamber cover 504 and innerchamber cover 505.

FIG. 6 illustrates a femoral head formation using a hollow fiberbioreactor 600 of the present invention. The fine mesh 606 is deformedto the shape of a femoral head 615. A permeable membrane 607 is linedinside of the fine mesh 606. A mixture of demineralized bone materials609 and cells 610 is added into the inner chamber 612 along with thehollow fibers 608 dispersed in the mixture of demineralized bonematerials 609 and cells 610. The ends of hollow fibers 608 are connectedto the two ports, inlet 601 and outlet 603, for nutrient delivery andwaste removal from the inner chamber 612. The permeable membrane 607 andfine mesh 606 are sealed to the end-plate 605 of the inner chamber 612.The end-plate 605 of the inner chamber 612 is connected to the end-plate604 of the outer chamber 611 through an annular groove: The nutrient isdelivered into the out chamber through the inlet port 614 and the wasteis removed from the outer chamber 611 through the outlet port 602.

The nutrient medium provided and the flow rate of this nutrient mediumwill vary depending on cell type added to the bioreactor, the packingdensity of the demineralized bone, presence/absence of a pre-added“extracellular matrix”, and numbers and kinds of hollow fibers containedwithin the inner vessel of the bioreactor. Nutrient flow will continueuntil such time as it has been previously determined that theappropriate matrix (structure) has been obtained. At this time, thebioreactor is aseptically dismantled and the bone or bone-like structureaseptically removed for further use.

The formed new bone can consist of a nonmineralized and nonload-bearingosteoid-like material that will mineralize when transplanted into aheterotopic or orthotopic site in a patient or a partially mineralizedand partially load-bearing osteoid material that will further mineralizewhen transplanted into a patient. Given time, it is also possible toproduce an almost completely mineralized bone-like tissue that will beload-bearing when implanted clinically.

In another aspect of the invention, the demineralized ground bone andbone-forming cells may form an extracellular matrix that is capable offorming bone when implanted in a patient. In this manner, thedemineralized bone and cells may be gelled in a viscous material andhave non-loading bearing implantable material that will form in vivosimilar to the in vitro bone-forming process described above.

The bone, bone-like tissue, and extracellular matrix made according tothe present invention is suitable for transplantation into a patient inneed thereof. As one having ordinary skill in the art would appreciate,the bone, bone-like material or tissue, and extracellular matrix can bemade into a desired shape that the body will remodel into theappropriate bone when implanted into a patient in some clinicalapplication. For example, as shown in FIGS. 7A and 7B, bone plugs formedin bioreactors of the present invention can have varying shapes andsizes. In particular, the bone plugs depicted in FIGS. 7A and 7B weregenerated after 4 weeks of incubation of ground demineralized boneparticles and human fibroblasts in the bioreactor.

Moreover, the bone, bone-like tissue, and extracellular matrix may befurther treated prior to implantation in manners known in the art. Forexample, these materials may be acellularized using known methods priorto implantation. Preferred methods of acellularization include, but arenot limited to, methods described in U.S. patent application Ser. Nos.09/528,371 and 09/660,422, which are hereby incorporated in theirentirety. The acellularized bone, bone-like tissue and extracellularmatrix is within the scope of the present invention. In addition, theseacellularized may be recellularized by known methods either in vitro orin vivo. Alternatively, any residual resorbable hollow fibers present inthe bone, bone-like tissue, or extracellular matrix may be removed usinghydrolytic enzymes, such as cellulase, chitinase, collagenase, elastase,proteases such as chymotrypsis, trypsin, ficin, papain and/or specificenzymes that are capable of degrading the polymers comprising theresorbable and non-resorbable hollow fibers and dialysis films. Otherknown methods of processing bone prior to implantation are furtherwithin the scope of the present invention.

EXAMPLES

The following examples are for purposes of illustration only and are notintended to limit the scope of the appended claims.

Example 1

Growth of New Bone Using a Sample Inner Vessel Consisting of DialysisMembrane Tubing in a Circulating Solution of Nutrient Solution

Dialysis tubes (Spectrum, Spectra/Por) made with different membrane poresizes (MWCO 10,000-25,000) and different material (regenerated celluloseor cellulose ester) were used for musculoskeletal bone tissueregeneration. The hydrogen peroxide in sterile dialysis tubes wasremoved and the tubes were soaked in tissue culture media for 1-2 hoursin order to remove all remnants of hydrogen peroxide. Demineralized bonematrices were weighed aseptically and hydrated with cell suspension(human dermal fibroblasts or human periosteal cells) in RPMI 1640 tissueculture medium. The DBM and cell mixtures were introduced into dialysistubes and the tubes were incubated in culture media containing 2% FBS,50 μg/ml L-ascorbic acid, 1 μM dexamethasone, and 50 mMbeta-glycerolphosphate. The dialysis system was incubated either understatic (that means the dialysis tubes are incubated in a mediacontainer), stirred dynamic (that means the dialysis tubes are incubatedin a media container which stays on stir plate to give constant mixingspeed), or fluid-flow dynamic (that means the dialysis tubes areincubated in a media flow chamber which controls the media flow rate fordialysis tubes by peristaltic pump) conditions. The culture media werereplaced by fresh media once a week to keep sufficient nutrients forcell growth and differentiation.

During various time of incubation (1-7 weeks), the culture media weretaken out from the containers for osteocalcin quantitation by ELISA, thetissues from the dialysis tubes were taken out for histology analysis,alkaline phosphatase quantitation, percentage of calcium quantitation,and double strand DNA quantitation. The samples of culture media weretaken out from bioreactor each week for osteocalcin quantification byELISA. FIG. 10 shows the time course of the bone protein, osteocalcin,levels for different cell seeding densities and identifies that theosteocalcin levels in the culture media increased significantly for thefirst 4 weeks and were more consistent after fourth week. Similarly, theosteocalcin levels normalized by the amount of DNA in the bone plugsgenerated in the bioreactor was also calculated based on the variouscell seeding densities and incubation time. As shown in FIG. 10, thelowest seeding density (0.5 million fibroblast cells per 100 mg of DBM)showed the highest osteocalcin level from second to fifth week ofincubation.

Various bone plugs produced according to this example were furtherexamined. Specifically, some of the bone plugs formed according to thisexample are depicted in FIGS. 7A and 7B, which indicates the variousshapes and sizes available to the person performing the invention.Additionally, FIG. 8 illustrates the bone plugs generated in thebioreactor that are subsequently freeze-dried. The shapes of these boneplugs reflect the shape of the deformable inner vessel of thebioreactor. FIGS. 11A-C and 12A-C illustrate the histological analysisof a bone plug generated in a bioreactor at 200× and 400× magnification,respectively. The “bone plug” generated in bioreactor was embedded andsectioned and the sections were stained with the Alizarin Red, H&E, andMasson's Trichrome methods. The Alizarin Red staining revealed thecalcium deposition in newly formed extracellular matrix. H&E stainingrevealed the changes in fibroblast morphology and new extra-cellularmatrix (ECM) production that appeared to be “osteoid” formation.Masson's Trichrome staining suggested that the newly formedextracellular matrix contained significant quantities of collagen.

FIGS. 13A-D illustrate the H&E staining of a bone plug generated in abioreactor and an analogous bone plug generated from heterotopicimplantation of DBM in a nude mouse (400× magnification). The new bonegrowth in a bioreactor (FIGS. 13A and 13B) of the present inventionafter 4 weeks incubation was compared to the new bone growth in a nudemouse (FIGS. 13C and 13D) 4 weeks after DBM implantation. The changes infibroblast morphology and new extracellular matrix production appearedon both samples.

FIGS. 14A-B illustrate the Mason's Trichrome staining of a bone pluggenerated in a bioreactor (FIG. 14A) and an analogous bone pluggenerated from heterotopic implantation of DBM in a nude mouse (FIG.14B) (400× magnification). Significant amounts of new extracellularmatrix were produced around cells and stained as collagen fibril forboth “bone plug” generated in a bioreactor and explants from a nudemouse.

FIG. 15 illustrates the alkaline phosphatase activity for bone plugsgenerated in a hollow fiber bioreactor with various cell seedingdensities. The group at a cell seeding density of 1×10⁷ human periostealcells per 500 mg of DBM showed significantly higher alkaline phosphataseactivity than other groups tested.

Example 2

Growth of New Bone Using a Prototypic Hollow-Fiber Containing Bioreactor

The bioreactor was constructed from glass tubing (inner diameter, 5 mm;length, 50 mm) and contained forty porous regenerated cellulose hollowfibers (outer diameter, 216 μm; inner diameter, 200 μm; MWCO of 18,000;Spectra/Por®; Spectrum Laboratories, Inc.; Laguna Hill, Calif.). Thehollow fibers were embedded in biomedical grade silicon rubber (NusilSilicone Technology, Carpenteria, Calif.).

To determine the optimal cell seeding density in the bioreactor system,human periosteal (HPO) cells were inoculated into the bioreactor atvarious cell density of 0.5×10⁶, 1×10⁶, 5×10⁶, and 1×10⁷ cells with DBM(1.5 cc or 500 mg). The culture medium used comprises Dulbecco'smodified Eagle's medium (DMEM) supplemented with antibiotics, ascorbicacid, beta-glycerophosphate, dexamethasone, and 2% fetal bovine serum(FBS). Two hundred and fifty ml of cell culture medium was recirculatedwith a medium flow rate of approximately 5 ml/min. After inoculation,the bioreactors were perfused using a peristaltic pump and maintained ina 5% CO₂/95% air incubator. After 5 days, the samples of cells with DBMwere removed and in vitro alkaline phosphatase assay was performed. FIG.16 represents the various cell seeding densities of HPO cells and theactivities of alkaline phosphatase from the in vitro alkalinephosphatase assay. These data demonstrate that HPO cells at a density of1×10⁷ cells have significantly higher alkaline phosphatase activitiesthan other groups with different cell seeding densities tested.

To study the growth of new bone or bone-like tissue using hollow-fiberbioreactor system, the bioreactor was inoculated with 1×10⁷ cells andDBM (1.5 cc or 500 mg) through either end into the extracapillary spaceof the bioreactor. Dulbecco's modified Eagle's medium (DMEM)supplemented with antibiotics, ascorbic acid, beta-glycerophosphate,dexamethasone, and 2% fetal bovine serum (FBS) was used as culturemedium throughout the experiments. Culture medium was changed weekly.Two hundred and fifty ml of cell culture medium was recirculated with amedium flow rate of approximately 5 ml/min. Diffusive nutrient supplyand removal of metabolic waste products across the membrane of hollowfiber was advanced by constantly recirculating culture medium throughthe system using a peristaltic pump maintained in a 5% CO₂ incubator.After 3 weeks, samples were taken from the bioreactors, fixed in neutralbuffered formalin, embedded in paraffin and sectioned. Sections werestained with Haematoxylin & Eosin. The results were illustrated in FIGS.16A-B showing H&E stained large cuboidal-shaped cells with deposition ofcollagen and organic bone matrix at 400× magnification.

Each of the patents and publications cited herein are incorporated byreference herein in their entirety. It will be apparent to one skilledin the art that various modifications can be made to the inventionwithout departing from the spirit or scope of the appended claims.

1. A method of growing new bone or bone tissue under in vitro cellculture conditions comprising, providing a bioreactor, grounddemineralized bone, nondemineralized bone, and bone-forming cells,wherein the nondemineralized bone comprises at least one ofnondemineralized bone fibers and nondemineralized bone particles,placing the ground demineralized bone, nondemineralized bone, and thebone-forming cells in the bioreactor, and providing a nutrient solutioncomprising nutrients and oxygen for the bone-forming cells, causing thenutrient solution to flow into, through, and out of the bioreactorcontaining the ground demineralized bone, nondemineralized bone, and thebone-forming cells to bring the flowing nutrient solution into contactwith the bone-forming cells, wherein nutrients and oxygen in the flowingnutrient solution are at sufficient concentrations to permitdifferentiation of the bone-forming cells into bone or bone tissue inthe bioreactor.
 2. The method of claim 1, wherein the bone-forming cellsare selected from the group consisting of stem cells, fibroblast cells,periosteal cells, chondrocytes, chondroblasts, osteocytes,pre-osteoblasts, osteoblasts and genetically modified cells.
 3. Themethod of claim 2, wherein the bone-forming cells are osteoblasts,chondrocytes, and chondroblasts.
 4. The method of claim 1, wherein theground demineralized bone is in the form of particles.
 5. The method ofclaim 4, wherein the ground demineralized bone is freeze-dried.
 6. Themethod of claim 5, wherein the ground demineralized bone is rehydratedprior to being placed in the bioreactor.
 7. The method of claim 5,wherein the ground demineralized bone is rehydrated after being placedin the bioreactor.
 8. The method of claim 4, wherein the grounddemineralized bone particles have an average size of about 50 microns toabout 4 mm.
 9. The method of claim 4, wherein the ground demineralizedbone particles haven an average size of about 250 microns to about 710microns.
 10. The method of claim 1, wherein the ground demineralizedbone is in the form of fibers.
 11. The method of claim 10, wherein thedemineralized ground bone fibers have a width of about 0.1 mm to about0.5 mm, a thickness of about 0.05 mm to about 0.5 mm, and a length ofabout 1 mm to about 500 mm.
 12. The method of claim 1, wherein thebone-forming cells synthesize and secrete bone or bone tissue matrix.13. The method of claim 12, wherein the bone matrix is viscous.
 14. Themethod of claim 1, further comprising adding collagen or hyaluronin tothe ground demineralized bone, nondemineralized bone, and bone-formingcells in the bioreactor.
 15. The method of claim 1, wherein the nutrientsolution comprises at least one of Dulbecc's Modified Eagle's Medium,fetal bovine serum, L-ascorbic acid, antibiotics, dexamethasone,beta-glycerolphosphate, glucose, glutamine, amino acid supplements,glutathione-ethyl ester, antioxidants, caspase inhibitors, cations, andanions.
 16. The method of claim 1, wherein the nutrient solution isintroduced into the bioreactor through resorbable hollow fibers.
 17. Themethod of claim 16, further comprising implanting the new bone or bonetissue into a recipient, and degrading the resorbable hollow fibersusing hydrolytic enzymes prior to implantation of the bone or bonetissue.
 18. The method of claim 16, further comprising introducinggrowth promoting factors through the resorbable hollow fibers into thebioreactor and degrading the resorbable hollow fibers thereby leavingchannels through the bone or bone tissue, wherein the new bone or bonetissue having the channels is implanted into a recipient.
 19. The methodof claim 18, further comprising delivery of growth factors through thechannels in the implanted bone or bone tissue to promote angiogenesis.20. The method of claim 1, wherein metabolic waste products are producedby the bone-forming cells in the bioreactor, and wherein the metabolicwaste products are removed from the bioreactor through resorbable hollowfibers.
 21. The method of claim 1, wherein at least one growth factor isintroduced into the bioreactor, and the ground demineralized bone, thenondemineralized bone, and the bone-forming cells are contacted with thegrowth factor.
 22. The method of claim 21, wherein the growth factor isa vascular endothelial growth factor.
 23. The method of claim 1, whereinthe ratio of mass of ground demineralized bone to nondemineralized boneis about 1:1 to about 20:1.
 24. The method of claim 1, furthercomprising implanting the new bone or bone tissue into a recipient,wherein the bone-forming cells are autogenic, allogenic, or xenogenicwith respect to the recipient.
 25. The method of claim 1, wherein thebone-forming cells are added to the bioreactor after the grounddemineralized bone and after the nutrient solution have been added tothe bioreactor.
 26. The method of claim 1, wherein the bone-formingcells are placed in the bioreactor prior to placing the grounddemineralized bone in the bioreactor.
 27. The method of claim 1, whereinat least one differentiation factor is introduced into the bioreactor,and the ground demineralized bone, the nondemineralized bone, and thebone-forming cells are contacted with the differentiation factor. 28.The method of claim 27, wherein the differentiation factor is a bonemorphogenetic protein.
 29. The method of claim 1, wherein at least onegrowth factor and one differentiation factor are introduced into thebioreactor, and the ground demineralized bone, the nondemineralizedbone, and the bone-forming cells are contacted with the growth factorand differentiation factor.
 30. The method of claim 29, wherein thegrowth fact is a vascular endothelial growth factor and thedifferentiation factor is a bone morphogenetic protein.